Project Summary Vpu enhances retrovirus particle release from the plasma membrane by inhibiting the potent cellular restriction factor BST2/tetherin. It has become clear that cellular trafficking pathways are the key to understanding the interplay between tetherin and Vpu. We have uncovered two major clues that to tetherin trafficking and Vpu action that form the basis for the first two Aims of this project. We have also found that tetherin contributes in a unique way to HIV replication in human macrophages, influencing the formation of the virus-containing compartment (VCC). The overall goals of this project are to define the mechanism of restriction of particle release by tetherin and the mechanism by which Vpu overcomes restriction through defining their interactions in the endosomal recycling complex (ERC). The first new discovery is that tetherin traffics throught the ERC, and can be quantitatively trapped in this compartment. Experiments in Aim 1 will define the Rab and Rab-related factors that specifically sort tetherin to the site of assembly and restriction on the plasma membrane, and will define a model for Vpu action that requires disruption of ERC trafficking. Experiments in Aim 2 follow up on a surprising finding that tetherin can rescue HIV-1 envelope onto matrix-mutant HIV particles that normally lack envelope, even restoring their infectivity. Defining the mechanism will uncover important facets of tetherin trafficking, including how the natural isoforms of tetherin differ in specifics of trafficking. Aim 3 will follow up on our discovery that tetherin is an important determinant of VCC formation, and focuses on the role of Siglec-1 and the role it plays in VCC formation and cell-cell transmission. Together these studies will continue a productive line of investigation into tetherin and Vpu biology and HIV replication in macrophages, and will facilitate new antiviral approaches and HIV cure strategies.